LIPOPROTEIN-APOPROTEI"
OF CHOLESTEROL-FED GUINEA PIGS
Changes in the Plasma Lipoprotein-Apoproteins of Guinea Pigs in Response to Dietary Cholesterol? Luke S. S. Guo,I Martha Meng, Robert L. Hamilton,$ and Rosemarie Ostwald*,s
ABSTRACT: The major apoproteins from four plasma lipo-
proteins were isolated from control and cholesterol-fed guinea pigs. Apoproteins were studied by column chromatography, polyacrylamide gel electrophoresis, and amino acid analysis. Dietary cholesterol altered the plasma apolipoproteins mainly by an enrichment in the content of arginine-rich polypeptide (ARP) in all density fractions. This protein had a similar
T h e response of guinea pigs to dietary cholesterol differs from that of other species by the appearance i n plasma of discoidal high density lipoproteins rich i n unesterified cholesterol, and by decreased levels of very low density lipoproteins (Sardet et al., 1972). A net accumulation of cholesterol i n liver, other tissues, and red cells is associated with pathological changes and with the formation of echinocytes. A fatal hemolytic anemia usually develops after IO- 14 weeks on a diet containing I % cholesterol (Ostwald and Shannon, 1964; Ostwald et al., 1977). We reported earlier on changes occurring in the lipid composition, electrophoretic mobilities, and ultrastructure of guinea pig plasma lipoproteins i n response to dietary cholesterol (Puppione et al., 1971; Sardet et al.. 1972). The present study presents data on the major apoprotein constituents of the lipoproteins from control and cholesterol-fed guinea pigs. We found that the major apoprotein appearing in the lipoproteins in response to dietary cholesterol is the arginine-rich polypeptide (ARP) and that its concentration may be proportional to that of plasma unesterified cholesterol. Ex perimen ta I Procedure Animals. Male albino guinea pigs weighing 200-250 g (Simonson Lab., Gilroy, Calif.) were fed one of two control diets (Purina chow with or without the addition of 5% cottonseed oil) or the Purina chow with 5% cottonseed oil diet supplemented with I % cholesterol (Matin and Ostwald, 1975). Cholesterol-fed animals were autopsied when the red cell count dropped below 3.5 X IO6 cells/mm3, usually after 10-12 weeks. Blood was collected in 0.1% NazEDTA' by heart puncture from animals fasted 15- 17 h . Red cells were immediately removed by low speed centrifugation and plasma was recentri-
' From the Department of hutritional Sciences, University of California, Berkeley, California 94720. and the Cardiovascular Research Institute. University of California, San Francisco. California 941 43. Receiced Augusi 24. 1977. Supported by the United States Public Health Service. National Institutes of Health Grant AM-8480, and Arteriosclerosis SCOR Grant HL-14237 of the National Heart, Lung and Blood Institute. A portion of this work has been presented at the Federation of American Societies for Experimental Biology (FASEB) Meeting. Atlantic City, April 1975. The major portion of this work was carried out while thc first author (L.S.S.G.)was holding a postdoctoral fellowship in the Department of Nutritional Sciences, University of California, Berkeley. Cardiovascular Research Institute. University of California, San Francisco, California 94143. Address correspondence to this author a t the Department of Nutritional Sciences, University of California. Berkeley. California 94720.
*
molecular weight (34 000), electrophoretic mobility, amino acid composition, and microheterogeneity as A R P reported in other mammalian species. The estimation of plasma concentration of A R P indicates a higher correlation coefficient with plasma unesterified cholesterol ( r = 0.98) compared with cholesterol esters ( r = 0.62).
fuged at 12 OOOg for 20 min at 4 "C to remove chylornicrons. Preparation of Plasma Lipoproteins. Densities of plasma and liporprotein fractions were adjusted by the addition of NaCI-NaBr solutions containing 0.01%NazEDTA and these solutions were centrifuged in a 40.3 rotor a t 40 000 rpm for 16-24 h a t 16 OC (Beckman Model L2-65B ultracentrifuge). Lipoproteins were recovered by standard techniques (Lindgren et ai., 1972). VLDL were isolated at 1.006 g/mL and refloated at the same density. LDLl ( d 1.01-1.05 g/mL) were floated at d 1.07 g/mL, sedimented at d 1.01 g/mL and refloated at d 1.05 g/mL. LDLz ( d 1.07-1.09 g/mL) and HDL ( d 1.09- 1.20 g/mL) were floated at d 1.21 g/mL, sedimented at d I .07 g / m L and refloated a t d 1.09 g / m L and d I .20 g/mL, respectively. Two density fractions of HDL were isolated from cholesterol-fed animals (HDLI, d I .07-1.09 g/mL, and HDL2, d I .09- I .20 g/mL) because two major H D L components of different flotation rates had been observed by analytical ultracentrifugation (Puppione et al., 1971). We found, as did Chapman et al. ( I 975), that the lipoproteins of d 1.07- 1.09 g / m L of control guinea pigs were typical of low density lipoproteins in their p electrophoretic mobility, content of Bapoprotein, size, shape, and chemical composition. I n contrast, this same density fraction from cholesterol-fed guinea pigs contained mainly discoidal high density lipoproteins with a electrophoretic mobility and little or no B-apoprotein. We therefore refer to the density fraction d 1.07-1.09 g / m L as control LDL2 and cholesterol H D L I , respectively. Isolated lipoprotein classes were examined by agarose gel electrophoresis (Noble, 1968), electron microscopy using negative staining, and immunological techniques. The results were essentially the same as those we previously reported (Puppione et al., 1971; Sardet et al., 1972). Lipoprotein Delipidation. Lipoprotein fractions at a concentration of 1-5 mg of protein/mL in a solution containing 0.1 5 M NaCI, 0.02% NaN3, and 0.04% EDTA, pH 7.6, were delipidated with 20 volumes of ethanol-ether (3:l) at -20 OC I Abbreviations used: ARP. arginine-rich polypeptide; Na2EDTA. disodium ethylenediaminetetraacetic acid: Tris, tris(hydroxymethy1)aminomethane; DEAE. diethylaminoethyl; VLDL. LDL, and HDL. very low density, low density, and high density lipoproteins; N a D o d S 0 ~aodium . dodecyl sulfate; T M U , tetramethylurea; apo-A, apo-B, and apo-C, apolipoproteins A, B, and C, respectively; BSA, bovine serum albumin; LCAT, lecithin-cholesterol acyl transferase.
BIOCHEMISTRY, VOL. 16, NO. 26, 1977
5807
G U O ET AL. R4T HUM4N I
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GP
- ARP 4-1 -A p o - C
